Antibodies to αvβ3 integrin

ABSTRACT

The invention relates to monoclonal antibodies to the αvβ3 integrin receptor known to be expressed in large amounts on the surface of osteoclasts and accordingly, associated with bone resorption. The disclosed monoclonal antibodies are believed to recognize unique epitopes on αvβ3 and are useful in the treatment of conditions associated with excessive bone resorption and/or in the inhibition of tumor cell growth.

CROSS REFERENCES

This application is a continuation of U.S. application Ser. No.08/307,844 filed 30 Sep. 1994, which application is a 371 ofPCT/US93/02987 filed Mar. 30, 1993, now U.S. Pat. No. 5,578,704 whichapplication is a continuation in part of U.S. application Ser. No.08/025,913 filed 3 Mar. 1993 (abandoned), which application is acontinuation of U.S. application Ser. No. 07/862,679 filed 3 Apr. 1992(abandoned), which applications are incorporated herein by reference andto which applications priority is claimed under 35 USC §120.

FIELD OF THE INVENTION

This application relates to hybrid cell lines (lymphocyte hybridomas)for the production of monoclonal antibodies to αvβ3 integrin, to suchhomogeneous antibodies, and to the use of such antibodies for diagnosticand therapeutic purposes.

BACKGROUND OF THE INVENTION

αvβ3 is a member of the integrin supergene family of cell-surfaceglycoprotein receptors that promote cellular adhesion. Each cell has aspecific repertoire of receptors that define its adhesive capabilities.The integrins are expressed as heterodimers of noncovalently associatedα and β subunits. According to the nomenclature proposed by Hynes, R. O.[Cell 48, 875-886 (1987)], the integrins can be divided into familieseach with a common β-subunit and a set of variable α-subunits known toassociate with the common β-subunit. The different α chains are denotedby the original cell type, by a subscript used by the originaldiscoverer, or, as in the case of the αvβ3 receptor, by the nature ofthe ligand (i.e. αv stands for a vitronectin receptor α-chain). Many,but not all, integrin receptors have been shown to interact withproteins via a tripeptide sequence, Arg-Gly-Asp (or RGD using the singleletter amino acid code), originally defined from studies of the cellbinding domains of fibronectin [Ruoslahti, E. and Pierschbachter, M. D.,Cell 44, 5170518 (1986); Ruoslahti, E. and Pierschbachter, M. D.,Science 238, 491-497 (1987)].

αvβ3 (also referred to as vitronectin receptor or VNR) is a member ofthe β3 integrin subfamily and is expressed on a variety of cells,including endothelial, melanoma, smooth muscle cells and, along withanother integrin α2β1 (VLA-2) (the receptor for Type I collagen andlaminin), on the surface of osteoclasts [Horton, M. A. and Davies, J.,J. Bone Min. Res. 4, 803-808 (1989); Davies, J. et al., J. Cell. Biol.109, 1817-1826(1989); Horton, M., Int. J. Exp. Pathol. 71 741-759(1990)]. αvβ3 mediates cell adhesion to vitronectin, fibrinogen,fibronectin, thrombospondin, osteopontin, bone sialo protein II and vonWillebrand factor.

Osteoclasts are the main type of bone cells involved in the resorptionof bone tissues. The resorption process involves the proliferation andchemotaxis of developing osteoclasts to the skeleton from hematopoieticsites migration of mature cells to sites of subsequent resorption,attachment of osteoclasts to bone substrate and the eventual formationof the polarized, functional mature and cells which are directlyinvolved in bone resorption. The αvβ3 integrin mediates adhesion ofosteoclasts to RGD sequence-containing bone matrix proteins.

Antibodies to αvβ3 are expected to be valuable diagnostic andtherapeutic tools in studying the biological role and thestructural/functional relationships of this integrin with its variousligands. In particular, monoclonal antibodies (Mabs) detecting uniqueepitopes on osteoclasts would be of great value in understanding of thedevelopment of osteoclasts. Even more importantly, neutralizing Mabsspecific for αvβ3 that inhibit the osteoclast binding to the bone matrixproteins have great potential as therapeutic agents useful in thetreatment of conditions associated with excessive bone resorption.

There are several monoclonal antibodies known in the art that bind tovarious epitopes on αvβ3. Immunizing with osteoclasts fromosteoclastomas (giant cell tumors of bone), Horton, M. A. et al. [CancerRes. 45, 5663-5669 (1985)] produced eleven mouse hybridomas secretingmonoclonal antibodies which bind to osteoclasts in normal human fetalbone and a variety of neoplastic and non-neoplastic bone lesions. One ofthese, designated 23C6, was subsequently shown to bind the αvβ3 complex,and was demonstrated to be able to disrupt osteoclast function [Horton,M. A. et al., Exp. Cell. Res. 195 368-375 (1991)]. Another monoclonalantibody, LM609 (produced in hybridoma LM609 ATCC HB 9537) disclosed inPCT Application Publication No. WO 89/05155 (published 15 Jun. 1989) andCheresh et al. J. Biol. Chem. 262:17703-17711 (1987) was also found tobind the αvβ3 complex and, due to its ability to inhibit the binding ofECr molecules present on the surface of tumor cells and blood vesselforming endothelial cells to vitronectin, fibrinogen and von Willebrandfactor, was proposed for therapeutic use as tumor growth inhibitor.Monoclonal antibody 13C2 (Horton, M. A. et al., Cancer Res. 1985, Supra)was shown to bind the αv portion of the αvβ3 molecule, whereas severalother monoclonal antibodies were reported to recognize the β3 portion[Nesbitt, S. et al., Epitope Analysis of the Vitronectin Receptor(CD51), In "Leukocyte Typing IV" White Cell Differentiation Antigens,Knapp, W. et al. (eds.) 1991, p. 1037]. The specific monoclonalantibodies variously reported in the art were shown to also bind toendothelial cells and various melanoma cell lines.

There is a need for high affinity monoclonal antibodies to the αvβ3integrin that are capable of effective inhibition of the binding of αvβ3expressing cells to αvβ3 ligands, such as vitronectin and fibronectin.

It would be further desirable to provide monoclonal antibodies to αvβ3that bind osteoclasts and optionally other cells known to express αvβ3.

It would be particularly desirable to provide monoclonal antibodies thatare effective inhibitors of αvβ3 binding to its ligands and whichspecifically bind osteoclasts without binding to other cells known toexpress αvβ3, i.e., which are more specific for the target integrin onosteoclasts.

SUMMARY OF THE INVENTION

The present invention is based on successful research involving theproduction and extensive characterization of monoclonal antibodies toαvβ3 integrin. Accordingly, the present invention is directed tomonoclonal antibodies, and derivatives thereof, which are capable ofrecognizing unique epitopes on αvβ3 and/or which exhibit high affinityfor αvβ3. The invention is specifically directed to monoclonalantibodies recognizing unique epitopes on the αvβ3 complex or the β3portion thereof. The invention is further directed to monoclonalantibodies effectively inhibiting the binding to vitronectin andfibrinogen of αvβ3 expressing cells. In a particularly important aspect,the invention is directed to monoclonal antibodies specifically bindingαvβ3 on osteoclasts but not other αvβ3 on other cells (e.g. melanomacells C32R, M-21, HA-A, HA-L and HT-144 and human umbilical veinendothelial cells).

In one aspect, the invention concerns an anti-αvβ3 monoclonal antibodythat is capable of: (1) inhibiting the binding of αvβ3 expressing cellsto fibrinogen, (2) binding osteoclasts, and (3) binding to substantiallythe same epitope recognized by any one of a monoclonal antibody selectedfrom the group consisting of 10C4.1.3, 9G2.1.3 and 9D4.9.1 or which hasan affinity for αvβ3 which is about equal to or greater than that of theforegoing three antibodies.

In another aspect, the invention concerns isolated nucleic acid encodingsuch antibodies, and hybridoma or recombinant cells producing suchantibodies.

In a further aspect, the invention concerns the therapeutic ordiagnostic use of such antibodies. The monoclonal antibodies of theinvention are useful as therapeutic agents, either by themselves or inconjunction with (chemo)therapeutic agents, to treat diseases orconditions that are characterized by excessive bone resorption and/or toinhibit tumor growth. The monoclonal antibodies of the invention alsoare useful in diagnostic and analytical assays for determining thepresence of αvβ3 on cells, cell typing and in histochemical tissuestaining.

These and further aspects will be apparent from the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the methods for generation of anti-αvβ3antibodies.

FIG. 2 depicts an immunoprecipitation of αvβ3 components using theantibodies of this invention, two positive controls (23C6 and 13C2) andan IgG negative control.

FIGS. 3A-F is a flow cytometry comparison of Mab 23C6 αvβ3 epitopescompared with the epitopes of several Mabs of this invention. FIG. 3Ashows staining of αvβ3 transformed cells with fluorescent labeled 23C6alone, FIG. 3B depicts staining with fluorescent labeled 23C6 incompetition with unlabeled 23C6, and FIGS. 3C, 3D, 3E and 3F illustratestaining with 4 Mabs of this invention in competition with labeled 23C6.

FIGS. 4A-B illustrates the ability of the Mabs to inhibit binding ofαvβ3 transformed 293 cells to fibrinogen (FIG. 4A) or vitronectin (FIG.4B). Interestingly, 23C6 could inhibit cell binding to fibrinogen, ascould the other Mabs. However, 9D4.9.1 demonstrated substantially higheraffinity than did any of the other Mabs tested. As shown in FIG. 4B,only Mabs 10C4.1.3 and 9D4.9.1 were able to substantially inhibit cellbinding to vitronectin, and again the latter exhibited higher affinitythan the other Mabs tested.

FIGS. 5A and 5B, respectively, depict the inhibition by various Mabs ofsoluble αvβ3 binding to fibrinogen and vitronectin. The results largelyparallel those shown in FIGS. 4A-B.

FIGS. 6A-B shows the immunoperoxidase histochemical staining of humanosteoclasts (multinucleated cells) from giant cell tumor of bone. 6A:Mab 10C4.1.3. 6B: IgG control antibody. Pictures were taken at 330×magnification.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions and General Methods

The term "monoclonal antibody" as used herein refers to a substantiallyhomogeneous population of antibodies, i.e., the individual antibodiescomprising the population are identical in specificity and affinityexcept for possible naturally occurring mutations that may be present inminor amounts. Note that a monoclonal antibody composition may containmore than one monoclonal antibody.

The monoclonal antibodies included within the scope of the inventioninclude hybrid and recombinant antibodies (e.g. "humanized" antibodies)regardless of species of origin or immunoglobulin class or subclassdesignation, as well as antibody fragments (e.g., Fab, F(ab')₂, and Fv),so long as they have the novel and unobvious characteristics of theantibodies described herein, in preferred embodiments being antibodiesthat are capable of binding to substantially the same epitope as onerecognized by monoclonal antibody 10C4.1.3, 9G2.1.3 or 9D4.9.1 and/orhave affinity for that epitope which is greater than or equal to theaffinity of 23C6 or 9D4.9.1.

Thus, the modifier "monoclonal" indicates the character of the antibodyas a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies of the invention may bemade using the hybridoma method first described by Kohler & Milsrein,Nature 256:495 (1975), or may be made by recombinant DNA methods. Forexample, see Cabilly, et al., U.S. Pat. No. 4,816,567; or Mage & Lamoyi,in Mognoclonal Antibody Production Techniques and Applications, pp.79-97(Marcel Dekker, Inc., New York, 1987).

In the hybridoma method, a mouse or other appropriate host animal isimmunized with αvβ3 integrin by subcutaneous, intraperitoneal, orintramuscular routes to elicit lymphocytes that produce or are capableof producing antibodies that will specifically bind to the protein usedfor immunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. Goding,Monoclonal Antibodies: Princicles and Practice, pp.59-103 (AcademicPress, 1986). Immunization with the extracellular domain of αvβ3(truncated αvβ3 not containing its transmembrane or cytoplasmic domains)as shown in the examples produced a surprisingly large population ofanti-αvβ3 antibodies and is believed in part to be responsible for theunique specificities and high affinities of several Mabs so identified.In addition, use of lymph node cells (rather than spleen or othertissue) as fusion partners was believed to be instrumental.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. U.S.A., and SP-2cells available from the American Type Culture Collection, Rockville,Md. U.S.A., or P3X63Ag8U.1 murine myeloma cells [Yelton et al., Curr.Top. Microbiol. Immunol. 81, 1 (1978)]. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies. Kozbor, J. Immunol. 133:3001 (1984).Brodeur, et al., Monoclonal Antibody Production Techniques andApplications, pp.51-63 (Marcel Dekker, Inc., New York, 1987). As noted,the hybridomas were prepared from lymph node fusions.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the individualchains or, preferably the αvβ3 complex. Preferably, the bindingspecificity is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA), or by FACS sorting. The monoclonalantibodies of the invention are those that bind to soluble or cell boundαvβ3 and which are neutralizing, as explained infra. Then thespecificity of binding αvβ3 on various cell types is determined, withthe objective being the identification of antibodies that do not bind toany other integrin than αvβ3 and, preferably, are able to discriminatebetween αvβ3 on melanoma tumor cells, endothelial cells and osteoclasts,i.e., are substantially specific for any one of such cell types.Substantial specificity in general means that the antibody is specificfor the candidate cell type at least to the degree of discriminationshown by Mab 10C4.1.3 for osteoclasts compared to any one of cell linesC32R, M-21, HA-A, HA-L, HT-63 or MG-63. Obviously this may be expressedin terms of the quantity of antibody that binds or in other conventionalmeasures. Finally, the screen optionally is narrowed to detectantibodies that bind to substantially the same epitope recognized byantibodies 10C4.1.3, 9G2.1.3 or 9D4.9.1 (as determined by competitionassays of the sort described infra for 23C6, except that the 3 Mabs ofthis invention will be employed as the labelled competitive agent todetermine epitope binding of the candidate). It should be kept in mindthat "same epitope" does not mean the exact amino acid or carbohydrateto which any of the three benchmark antibodies bind, as may bedetermined for example by epitope mapping using alanine scanned variantsof αvβ3. "Same epitope" means the αvβ3 domain which is blocked by thebinding to αvβ3 of one of the native benchmark antibodies in intactform. Of course, "same epitope" includes the αvβ3 domain residues orcarbohydrate that structurally interacts or binds to the benchmark CDRs.

In a preferred embodiment of the invention, the monoclonal antibody willhave an affinity which is greater than that of 23C6 and preferably isequal or greater than that of 9D4.9.1, as determined, for example, bythe Scatchard analysis of Munson & Pollard, Anal. Biochem. 107:220(1980).

The term "neutralizing antibody" as used herein refers to a monoclonalantibody that is capable of substantially inhibiting or eliminating abiological activity of αvβ3. Typically a neutralizing antibody willinhibit binding of αvβ3 to a cell matrix ligand such as vitronectin orfibrinogen to a degree equal to or greater than Mab 23C6, and preferablyequal to or greater than Mabs 9D4.9.1, 10C4.1.3 or 9G2.1.3.

After hybridoma cells are identified that produce neutralizingantibodies of the desired specificity and affinity, the clones typicallyare subcloned by limiting dilution procedures and grown by standardmethods. Goding, Monoclonal Antibodies: Principles and Practice,pp.59-104 (Academic Press, 1986). Suitable culture media for thispurpose include, for example, Dulbecco's Modified Eagle's Medium orRPMI-1640 medium. In addition, the hybridoma cells may be grown in vivoas ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA is ligated into expression or cloning vectors, whichare then transfected into host cells such as simian COS cells, ChineseHamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein. The transformant cells are cultured toobtain the synthesis of monoclonal antibodies in the recombinant hostcell culture.

The DNA optionally is modified in order to change the character of theimmunoglobulin produced by its expression. Immunoglobulin variants arewell known. For example, chimeric antibodies are made by substitutingthe coding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Cabilly et al. op cit, orMorrison, et al., Proc. Nat. Acad. Sci. 81:6851 (1984). In addition, theFc domain chosen is any of IgA, IgD, IgE, IgG-1, -2, -3 or -4, or IgM.The Fc domain optionally is capable of effector functions such ascomplement binding.

Humanized forms of the murine antibodies are made by substituting thecomplementarity determining regions of the mouse antibody into a humanframework domain, e.g., see PCT Pub. No. WO92/22653, published 23 Dec.1992. In some embodiments, selected murine framework residues also aresubstituted into the human recipient immunoglobulin.

Fusions of the immunoglobulins of this invention and cytotoxic moietiesare made, for example, by ligating to the immunoglobulin coding sequenceall or part of the coding sequence for a cytotoxic non-immunoglobulinpolypeptide. Such non-immunoglobulin polypeptides include polypeptidetoxins such as ricin, diphtheria toxin, or Pseudomonas exotoxin.

Also, the conjugates can be prepared by in vitro methods. For example,immunotoxins may be constructed using a disulfide exchange reaction orby forming a thioether bond between the immunoglobulin and the toxinpolypeptide. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate. In addition, otherfusions are readily produced by similar recombinant methods. Suitablefusion partners for the immunoglobulin of this invention include viralsequences, cellular receptors such as the T-cell receptor, cytokinessuch as TNF, interferons, or interleukins, and other biologically orimmunologically active polypeptides. Typically such non-immunoglobulinfusion polypeptides are substituted for the constant domains of anantibody of the invention. Alternatively, they are substituted for thevariable domains of one antigen-combining site of an antibody of theinvention.

Substitution of the Fr or CDRs of an antibody having specificity for anon αvβ3 antigen will create a chimeric bivalent antibody comprising oneantigen-combining site having specificity for αvβ3 and anotherantigen-combining site having specificity for a different antigen. Insuch embodiments, the light chain is deleted and the Fv of the heavychain is substituted with the desired polypeptide. These antibodies aretermed bivalent or polyvalent, depending upon the number ofimmunoglobulin "arms" possessed by the Fc domain employed (IgMs will bepolyvalent). Aside from the nonimmunoglobulins mentioned above, theantibody also is rendered multivalent by recombination of antibodieshaving more than one specificity. For instance, the antibody in someembodiments is capable of binding αvβ3 as described elsewhere herein butis also capable of binding a T-cell determinant such as CD3, CD4, CD8,CD18, CD11a, CD11b or CD11c. These other antibodies are well known. Themultispecific, multivalent antibodies are made by cotransforming a cellwith DNA encoding the heavy and light chains of both antibodies and theproportion of expressed antibodies having the desired structurerecovered by immunoaffinity chromatography or the like. Alternatively,such antibodies are made from monovalent antibodies which are recombinedin vitro in conventional fashion.

Monovalent antibodies also are made by techniques that are conventionalper se. Recombinant expression of light chain and a modified heavy chainis suitable. The heavy chain is truncated generally at any point in theFc region so as to prevent heavy chain crosslinking. Alternatively, therelevant cysteines are substituted with another residue or deleted so asto prevent crosslinking. In vitro methods also are used to producemonovalent antibodies, e.g., Fab fragments are prepared by enzymaticcleavage of intact antibody.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as all, ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, afluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; radioactive isotopic labels,such as, e.g., ¹²⁵ I, ³² P, ¹⁴ C, technicium, or ³ H, or an enzyme, suchas alkaline phosphatase, betagalactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniaues, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be αvβ3 or an immunologically reactive portion thereof) tocompete with the test sample analyte (αvβ3) for binding with a limitedamount of antibody. The amount of αvβ3 in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

The antibodies of the invention also are useful for in vivo imaging,wherein an antibody labeled with a detectable moiety such as aradio-opaque agent or radioisotope is administered to a host, preferablyinto the bloodstream, and the presence and location of the labeledantibody in the host is assayed. This imaging technique is useful in thestaging and treatment of neoplasma or bone disorders. The antibody maybe labeled with any moiety that is detectable in a host, whether bynuclear magnetic resonance, radiology, or other detection means known inthe art.

The neutralizing antibodies of the invention are especially useful intherapeutic applications, to prevent or treat unwanted bone resorption,or tumor cell growth or metastasis. Obviously, Mabs of the 10C4.1.3 typeare not useful for treating or in vivo imaging of tumors of the sametype described in Table 2 infra since they do not bind to αvβ3 found onsuch cells. Instead these Mabs are especially useful of treatingconditions of bone resorption or degradation, for example as found inosteoporosis or resulting from PTHrP over-expression by some tumors.

For therapeutic applications, the antibodies of the invention areadministered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form. They are administered intravenously as a bolusor by continuous infusion over a period of time, by intramuscular,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. When the antibody possesses the suitableactivity it is also suitably administered by intratumoral, peritumoral,intralesional, or perifesional routes, to exert local as well assystemic therapeutic effects.

Such dosage forms encompass pharmaceutically acceptable carriers thatare inherently nontoxic and nontherapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffers such as phosphate orglycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts, or electrolytes such asprotamine sulfate, sodium chloride, metal salts, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, cellulosic polymers, andpolyethylene glycol. Carriers for topical or gel-based forms of antibodyinclude polysaccharides such as sodium carboxymethylcellulose ormethylcellulose, polyvinylpyrrolidone, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols. Conventional depot forms include, for example,microcapsules, nano-capsules, liposomes, plasters, sublingual tablets,and polymer matrices such as polylactide:polyglycolide copolymers. Whenpresent in an aqueous dosage form, rather than being lyophilized, theantibody typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibodiesare administered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

Depending on the type and severity of the disease, about 0.015 to 15 mgof antibody/Kg of patient weight is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and arenot excluded herefrom.

According to another embodiment of the invention, the effectiveness ofthe antibody in preventing or treating disease may be improved byadministering the antibody serially or in combination with another agentthat is effective for the same clinical objective, such as anotherantibody directed against a different epitope than the principalantibody, or one or more conventional therapeutic agents known for theintended therapeutic indication, e.g. prevention or treatment ofconditions associated with excessive bone resorption such as,osteoporosis or inhibition of tumor cell growth or metastasis.

The antibodies of the invention also are useful as affinity purificationagents. In this process, the antibodies against αvβ3 are immobilized ona suitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the αvβ3 to be purified, and thereafter the support iswashed with a suitable solvent that will remove substantially all thematerial in the sample except the αvβ3, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent, such as glycine buffer, pH 5.0, that will release theαvβ3 from the antibody.

The following examples are offered by way of illustration only and arenot intended to limit the invention in any manner.

EXAMPLE 1

A. Generation of Mabs specific for human αvβ3

To produce Mabs specific for αvβ3 integrin, Balb/c mice were immunizedwith αvβ3 integrin purified from 293-15D cell line expressing αvβ3complex which were generated by transfecting 293 cells (ATCC CRL1573)with DNAs prepared from PMNCV vector expressing αv or β3 plus DNA codingfor Neomycin resistance gene. αvβ3 was purified from an NP 40 celllysate of 293-15D cells by using a lentil lectin column. The purity ofthe αvβ3 prepared was then confirmed by isoelectrophoresis. Mice wereimmunized into foot pads once with 5 μg of αvβ3 emulsified in MPL/TDMadjuvant (Ribi Immunochem. Research Inc., Hamilton, Mont.) and thensubsequently six times with 5 mg of αvβ3 immersified in MPL/TDM adjuvantat 2 week intervals. Three days after the last immunization, lymph nodecells from these mice were fused with P3X63Ag8U.1 myeloma cells (Yelton,et al., Curr. Top. Microbiol. Immunol. 81:1, 1978) using 35%polyethylene glycol as described (Yarmush et al, Proc. Nat. Aced. Sci.77:2899, 1980). The remainder of the process is depicted in FIG. 1.Hybridoma cell lines were selected for anti-αvβ3 antibody production bytheir ability to bind soluble αvβ3 by ELISA and to bind cell linesexpressing various integrins by Flow microfluorometry analysis usingFACSCAN (Becton Dickinson FACS systems, Mountain View, Calif.). Isotypes(Table 1) of these positive Mabs were determined by ELISA using Isotypespecific alkaline phosphstase-conjugated goat anti-mouse immunoglobulin(Harlow and Lane, Antibodies: A Laboratory Manual. p.597, Cold SpringHarbor Laboratory, 1988).

                  TABLE 1    ______________________________________    Characteristics of Mabs    Cell Line            Isotype   Immunoblot Immune ppt                                          Epitope    ______________________________________    9D4.9.1 IgG1, K   -          +        β3    9G2.1.3 IgG2a, K  -          +        ανβ3    10C4.1.3            IgG1, K   -          +        ανβ3    ______________________________________

Positive hybridoma cell lines were subcloned twice by limiting dilutiontechnique.

B. Immune Precipitation of αvβ3 complex with Mabs

293-15D transfectants grown F12/DMEM medium with 10% FCS were harvestedby treatment with EDTA and biotinylated by using NHS-LC-Biotin. Cells(5×106 cells/ml) were incubated with 1 μg/ml of NIH-LS-Biotin for 1 hourat room temperature. The unbound biotin was then removed by washing in0.05 mM tris buffer containing 0.05M NaCl, 1 mM CaCl₂ and 1 mM MgCl₂(cell wash buffer). Cells were lysed by treatment with 1% NP-40 and celldebris were removed by microcentrifugation for 10 min. The supernatantwas used for the immunoprecipitation. Fifty μl of Protein-G in 0.05Mtris buffer containing 0.5M NaCl and 0.1% Tween-20 (IP wash buffer) wereincubated with 100 μl of Mabs (100 μg/ml) for 30 min at roomtemperature. After washing twice in IP wash buffer, nonspecific bindingsites on Protein-G were blocked with 1% BSA for 1 hr. at roomtemperature, washed twice and incubated with the supernatant containingbiotinylated membrane proteins for 1 hr at room temperature. The complexwas washed six times, reduced in SDS PAGE-sample buffer containing 2-MEby boiling and analyzed by electrophoresis using 12% SDS polyacrylamidegel. FIG. 2 depicts the results.

EXAMPLE 2 Cell culture and Immunofluorescence staining of varioustissues

Transfected cells and tumor cells grown in F12/DMEM medium (1:1 o/omixture) containing 10% FCS, glutamine and antibiotics were washed threetimes in the cell sorter buffer (CSB, PBS containing 1% FCS and 0.01%NAN3) by centrifugation at 1,000 rpm for 5 min and resuspended to be4×10⁸ cells/ml in CSB. Twenty five μl of cells were added into a 96 wellU-bottom plate and incubated with 100 μl of antibodies for 30 min onice. At the end of the incubation, calls were washed twice in CSB andthe Mab bound onto cells were detected by incubating cells with FITCconjugated goat anti-mouse 1 g antibodies for 30 min on ice. Cells werewashed twice in CSB, resuspended in 0.5 ml of CBS, and analyzed by Flowmicrofluorometry as described (Loken, et al. Ann. N.Y. Acad. Sci.254:163-; Miller, et al., Rev. Sci. Instrum, 49:1137-9, 1978). Theresults are shown in Table 2.

                                      TABLE 2    __________________________________________________________________________    Flow microfluorometry analysis of mabs with various cells                     2B4.12.1                           9C9.11.11                                 9C9.12.1                                       9D4.9.1                                            9G2.1.3                                                 10C4.1.3                                                       23C6    __________________________________________________________________________    Transfected Cells    293              -     -     -     -    -    -     -    293-52B (ανβ1)                     -     -     -     -    -    -     -    293-15D (ανβ3)                     +++   +++   +++   +++  +++  +++   +++    292-CLB (IIb-IIIa)                     +++   +++   +++   +++  +    -     ++    Human Melanomas    C32R             ++    ++    ++    ++   ++   -     ++    M-21             ++    ++    ++    ++   ++   +     ++    HA-A             ++    ++    ++    ++   ++   -     ++    HA-L             ++    ++    ++    ++   ++   -     ++    HT-144           ++    ++    ++    ++   ++   -     ++    Osteosarcoma (MG-63)                     +     +     +     +    +    -     -    Human Umbilical Vein Endothelium                     +     +     +     +    ++   -     +    Human Osteoclasts*                     +++   +++   +++   +++  +++  +++   +++    __________________________________________________________________________     FACS Results Graded: - (Mean FL Channel 0-10), + (Mean FL Channel 10-100)     ++ (Mean FL Channel 100-1000), +++ (Mean FL Channel >1,000)     *Tissue Frozen Section (Osteoclastoma), tissue section results graded: +     (weak), ++ (moderate), +++ (strong).

Table 2 depicts the determination of the portion of αvβ3 recognized bythese Mabs by FACS analysis of transfected cells expressing differentintegrins as well as by ELISA using soluble integrin proteins. Mab9G2.1.3 bound strongly to 293-15D expressing αvβ3 and very weakly to293-CLB expressing IIb-IIIa, but not 293-52B expressing αvβ3. Mab10C4.1.3 only bound to 293-15D but not others. Therefore, it wasconcluded that these two Mabs (9G2.1.3 and 10C4.1.3) recognized αvβ3. Incontrast, Mab 9D4.9.1 strongly bound to both 293-15D as well as 293-CLBbut not 293-52B. Thus, the Mab 9D4.9.1 was concluded to the bind β3portion of αvβ3.

Table 2 also shows that Mab 9C9.11.11 bound to osteoclasts, humanendothelial cells and various melanoma cells. This staining pattern wassimilar to that of 23C6. In contrast, Mab 10C4.1.3 recognized onlyoateoclasts, suggesting a surprising and very narrow specificity. Thestaining pattern of Mabs 9D4.9.1 and 9G2.1.3 for human melanoma tumorcells, glioma cells and normal endothelial cells were similar to that ofMab 23C6: these Mabs recognized various human melanoma cells, andosteoclasts strongly, and human osteosarcoma MG-63 cells and humanendothelial cells weakly. In contrast Mab 10C4.1.3 showed a strongbinding to human osteoclasts and a weak binding to one of the humanmyeloma cells, M-21, but no binding to other cells. These resultssuggest that 10C4.1.3 recognizes an epitope unique to human osteoclasts.

EXAMPLE 3 Determination of epitopes by competitive Immunofluorescencestaining

αvβ3 transfected 293-15D cells (1×10⁵ cells/100 μl) were incubated with100 μl of the first purified Mab for 30 min on ice, washed twice andincubated with the second Mab, FITC conjugated Mab 23C6 for 30 min. Atthe and of the incubation, cells were washed twice in CSB andresuspended in 0.5 ml of CBS and the level of FITC conjugated Mab 23C6binding on 293-15D cells was examined by flow microfluorometry(FACSCAN), The results are depicted in FIG. 3. Panels are as follows:

(3A) NONE+FL-23C6 (shaded area represents unstained cells)

(3B) 23C6+FL-23C6

(3C) 9D4.9.1+FL-23C6

(3D) 9G2.1.3+FL-23C6

(3E) 10C4.1.3+FL-23C6

(3F) IgG+FL-23C6

Several Mabs which recognize the α-chain of αvβ3 designated as CD51, orβ-chain of vitronectin receptors, CD61, have been described (Nesbitt etal, 1991, in "Leukocyte typing IV, p1037). More recent study (Horton,Int. J. Exp. Pathol., 71:741 [1990]) showed that Mab 23C6 and LM609,which were grouped as Mabs recognizing the β epitope of CD51, mayrecognize the intact αvβ3 complex. Thus to confirm that the Mabs bindingto the αvβ3 complex recognize different epitopes from that of 23C6, thestaining of 293-15D with fluoresceinated 23C6 was examined in thepresence of 100 fold higher level of unlabeled Mabs and analyzed byFACSCAN. The results in FIG. 3 showed that Mabs 9D4.9.1 and 10C4.1.3 didnot interfere at all with the binding of fluoresceinated-23C6 to 15Dcells. In the same experiment, the same amount of unlabeled 23C6completely inhibited the binding of F-23C6 while the irrelevant controlMab did not have any effect. Thus it was confirmed that at least thesetwo Mabs indeed recognize epitopes different from those recognized by23C6. In contrast, Mab 9G2.1.3 at high concentration, has someinhibitory effect on the F-23C6 binding but could not completely blockthe F-23C6 binding. Thus it is concluded that Mab 9G2.1.3 recognizes adifferent epitope from the one recognized by F-23C6, but these epitopesappear to be closely orientated. Since it has been reported that LM 609(Cheresh, et al., J. Biol. Chem., 262:17703-17711 [1987]) recognizes thesame epitope is 23C6 it is concluded that our Mabs recognized differentepitopes from the one recognized by Mab LM 609.

EXAMPLE 4 Inhibition of the binding of 293-15D cells to ligands(fibrinogen and vitronectin) by Mabs

Microplates (NUNC, Breakapart C8 Maxi Sorp) were coated with 100 μl/wellof 10 μg/ml of fibrinogen or vitronectin overnight at 4° C. After beingwashed three times in PBS, plates were blocked with 1% BSA in PBS for 1hr and then were incubated with various concentrations of Mabs for 30min followed by the addition of 100 ml of ⁵¹ Cr labeled 293-15D cells.Plates were centrifuged at 600 rpm for 2 min and incubated for 90 min at37° C. At the and of the incubation, plates were washed three times and⁵¹ Cr labeled 293-15D cells bound to the ligand were counted by a gammacounter. ⁵¹ Cr labeled 293-15D transfectants expressing αvβ3 wereprepared as follows. Cells were grown in F12/DMEM medium containing 10%FCS, 0.1% glucose and 2 mM glutamine for 40 hr, were harvested bytreatment with 10 mM EDTA in PBS for 2 min, washed twice in PBS andresuspended to be 5×10⁷ cells/ml in culture medium without FCS. 0.5 mlof 293-15D cells were then incubated with 250 mCi ⁵¹ Cr and for 1 hr at37° C. At the and of the incubation, excess unbound ⁵¹ Cr was removed bywashing three times in F12/DMEM medium and resuspended to be 6×10⁵cell/ml in culture medium without FCS. FIGS. 4A-B depicts the results.

The top panel of FIG. 4 shows the binding of ⁵¹ Cr-293 15 D tofibrinogen coated wells in the presence of various concentration ofMabs. All three Mabs inhibit the binding of ⁵¹ Cr-293 15 D to fibrinogenvery effectively. The strongest inhibition was shown with Mab 9D4.9.1.The bottom panel shows the binding of ⁵¹ Cr-293 15 D to vitronectincoated wells. Under the conditions tested, 9D4.9.1 and 10C4.1.3 couldinhibit this interaction but 9G2.1.3 and 23C6 showed a very weakinhibition, if any. In general, it was harder to inhibit the interactionbetween αvβ3 transfected cells to vitronectin than the interactionbetween αvβ3 transfected cells to fibrinogen.

EXAMPLE 6 Inhibition of αvβ3 integrin binding to Fibringen andVitronectin by monoclonel antibodies

Microtiter plates were coated with 100 μl/well of 10 μg/ml of purifiedfibrinogen or vitronectin, overnight at 4° C. After washing three timesin PBS, the plates were blocked with 1% BSA for 1 hour at roomtemperature. After washing the plate in PBS, ⁵¹ Cr-293-15D cellspreincubated with various concentrations of Mabs for 30 minutes on icewere transferred to the ligand coated plate. The plates were thencentrifuged at 600 rpm for 2 minutes and incubated for 90 minutes at 37°C. At the and of the incubation, the plates were washed three times inPBS and the ⁵¹ Cr labeled 293-15D cells bound to the ligand were countedby a gamma counter. The results, which parallel those in Example 4, aredepicted in FIGS. 5a and 5b.

EXAMPLE 7 Histochemical staining of frozen sections of human tissues andbone imprints

Frozen sections of human osteoclastoma tumors and bone imprints fromhuman fetal limb bones (14 weeks gestation), newborn rabbit and ratbone, embryonic chick bone and adult red deer antler, and from thefollowing tissues of adult human origin (liver, kidney, pancreas, colon,ileum, heart, lung, thymus, tonsil, spleen, placenta, skin, uterinecervix, umbilical cord, breast carcinoma, malignant melanoma, smears ofperipheral blood and bone marrow mononuclear cells) were prepared asdescribed [Horton, M. A. et al., Cancer Res. 45, 5663-5669 (1985)];these were air-dried, fixed in acetone for 10 minutes at roomtemperature and stored at -20° C. until use. Slides were brought to roomtemperature, rehydrated in PBS then incubated with 150 μl of 1% FCS/PBScontaining 1 μg of purified Mabs for one hour. After washing in 1%FCS/PBS the slides were incubated sequentially with biotinylatedanti-mouse 1 g and then with avidin-biotin-horseradish peroxidasecomplex at the manufacturer/s recommended dilutions (Vector Lab,Burlingame, Calif.) for one hour. After further washes, the boundperoxidase was developed in 0.1 mg/ml diaminobenzidinetetrahydrochloride containing 0.07% H₂ O₂, (Organon Teknina Corp.Durham, N.C.) in pBS and counter stained with 0.5% methyl green for 5minutes. The slides were then dehydrated in graded alcohols, thencleared in xylene and mounted in permountant (Fisher Scientific Co., SanFrancisco, Calif.) for microscopy.

FIG. 6 shows clear membrane staining of multinucleated osteoclasts fromhuman bone imprints and frozen sections of giant cell tumor of boneusing Mab 10C4.1.3; a control Mab did not show staining. Mabs 9D4.9.1and 9G2.1.3 showed a similar staining pattern to Mab 10C4.1.3. None ofthe Mabs recognized osteoclast vitronectin receptor in conventionalformalin-fixed and paraffin-embedded tissue sections.

We have examined the binding of these Mabs to osteoclasts present inbone imprints from rat, rabbit, chicken and deer in comparison to human.

Bone imprints were stained with mAbs followed by F-goat anti-mouse IgG.The level of fluorescence staining was examined by fluorescentmicroscopy and graded as weak (+), moderate (++) and strong (+++) orabsent (-).

                  TABLE 3    ______________________________________    Determination of the mAb binding to osteoclasts from various species            mAb    Species   9D4.9.1 9G2.1.3     10C4.1.3                                         23C6    ______________________________________    Human     +++     +++         +++    +++    Rat       -       -           -      -    Rabbit    -       +++         -      +++    Chicken   -       +++         -      +++    Deer      -       ++          -      +++    ______________________________________

Mab 9G2.1.3 recognizes osteoclasts from rabbit, chicken, deer inaddition to human; this distribution is similar to that seen with 23C6(Horton et al., 1985, op. cit.). In contrast, Mabs 9D4.9.1 and 10C4.1.3only recognized human osteoclasts; to date, no αvβ3 complex-specificMabs have shown such species selectivity.

The distribution of the antigens recognized by the three Mabs wasanalyzed by immunohistochemistry on frozen sections from theabove-listed tissues of normal adult and fetal origin (data not shown).Mab 9D4.9.1 stained platelets (and megakaryocytes in bone marrow)intensely in all tissues studied. In addition, vascular endothelium wasstained, variably and weakly, in all tissues. Mab 9G2.1.3 also stainedvascular endothelium, but failed to react with platelets andmegakaryocytes. Both antibodies attained kidney (glomerulus, tubules(,hepatic sinusoids, colonic and ileal smooth muscle, placenta (cyto- andsyncytiotrophoblasts) and neoplastic melanocytes in malignant melanoma.In contrast, Mab 10C4.1.3 failed to stain or gave a much weaker reactionin tissues recognized by Mab 9G2.1.3. For example, Mab 10C4.1.3 did notstain intestinal smooth muscle or placenta.

The antigenic specificities recognized by these Mabs were furtherinvestigated by examining their binding to various cell lines includinghuman melanoma tumor cells, osteosarcoma cells and normal humanumbilical vein endothelial cells (HUVEC) by flow microfluorometry. Mabs9D4.9.1 and 9G2.1.3 bound strongly to various human melanoma cells andless to MG-63 human osteosarcoma cells and HUVEC. In contrast, Mab10C4.1.3 bound weakly to only one of the human melanoma cell lines,M-21. These results further confirm that 10C4.1.3 recognizes a novelantigenic epitope.

The following antibody producing hybridomas have been deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,U.S.A. (ATCC):

    ______________________________________    Antibody     ATCC Dep. No.                             Deposit Date    ______________________________________    10C4.1.3     HB 11029    28 April 1992    9G2.1.3      HB 11030    28 April 1992    9D4.9.1      HB 11031    28 April 1992    ______________________________________

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromthe date of deposit or for the enforceable life of the patent or for aperiod of five years after the last request or for the effective life ofthe patent, whichever is longer. The organisms will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 USC §122 and the Commissioner's rules pursuant thereto (including37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if the cultureon deposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of the depositedstrain is not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

In respect of those designations in which a European patent is sought, asample of the deposited microorganism will be made available until thepublication of the mention of the grant of the European patent or untilthe date on which the application has been refused or withdrawn or isdeemed to be withdrawn, only by the issue of such a sample to an expertnominated by the person requesting the sample. (Rule 28(4) EPC)

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments without diverting from theoverall concept of the invention. All such modifications are intended tobe within the scope of the invention.

We claim:
 1. A composition comprising an anti-αvβ3 integrin monoclonalantibody 9G2.1.3, which monoclonal antibody is secreted by a hybridomacell line deposited under American Type Culture Collection AccessionNumber ATCC HB
 11030. 2. The monoclonal antibody composition of claim 1capable of (1) binding osteoclasts and (2) inhibiting the binding ofαvβ3 integrin expressing cells to fibrinogen.
 3. The monoclonal antibodycomposition of claim 2 which binds to the αvβ3 integrin complex.
 4. Ahybridoma cell which produces the monoclonal antibody of claim
 1. 5. Ahybridoma cell line deposited under American Type Culture CollectionAccession Number ATCC HB
 11030. 6. A monoclonal antibody produced by thehybridoma cell of claim
 5. 7. An immunoassay for the detection of αvβ3integrin comprising the steps of:(a) contacting a test sample with themonoclonal antibody of claim 1, and (b) determining the amount of αvβ3integrin in the test sample that is bound to the monoclonal antibody. 8.The monoclonal antibody of claim 1 which is labelled with a detectablemarker.
 9. The monoclonal antibody of claim 1 which is covalently bondedto a water insoluble matrix.
 10. The monoclonal antibody of claim 1which comprises an Fc domain having an immunoglobulin effector function.11. The monoclonal antibody of claim 1 which is monovalent.
 12. Themonoclonal antibody of claim 1 which comprises an Fc domain of IgA, IgD,IgE, IgG or IgM.
 13. The monoclonal antibody of claim 1 wherein theantibody is bound to a cytotoxin.